This invention relates to an enzyme electrode for assaying maltose, and particularly to a stable maltose sensor with a long life.
Maltose is a dimer of glucose and is produced when polysaccharides such as starch, etc. are hydrolyzed by .alpha.-amylase (which will be hereinafter referred to as "amylase"), etc. Thus, the amount of amylase can be determined indirectly by allowing amylase to act on a system containing an excess and a predetermined amount of a substrate, and measuring the amount of maltose thus produced.
Amylase is an enzyme capable of decomposing polysaccharides such as starch, dextrin, glycogen, pectin, etc. as a substrate into maltose, and exists in organs of animals including human being, plants and microorganisms. Diagnosis of various diseases can be made by quantitative analysis of amylase in biological fluids such as blood, etc., and thus the quantitative determination of amylase has been recently regarded as particularly important.
Heretofore available methods for quantitative analysis of amylase include (1) an amyloclastic method for tracing gradual decomposition of starch by amylase according to iodine-starch reaction, (2) a saccharogenic method for measuring the reducibility of maltose produced through decomposition by amylase, (3) a chromogenic substrate method for colorimetry of soluble pigments freed from insoluble colored starch, as crosslinked with pigments, as a substrate under the action of amylase, etc. Particularly when the sample is a biological fluid, these methods have a drawback of poor assaying accuracy, because various substances contained in the biological fluid, for example, urea, ureic acid, protein, sugars, vitamin C, etc. act as assay-interferring substances, and also have further drawbacks of complicated assaying operation and prolonged assay time.
To overcome these drawbacks, enzymatic methods have been recently developed, which include (4) a maltose phosphorylase method comprising decomposing maltose, which has been produced from soluble starch as a substrate by .alpha.-amylase, by maltophosphorylase and ultimately measuring the amount of NADH (reduced nicotinamid adenin dinucleotide) after further three enzyme reaction stages each using .beta.-phosphoglucomutase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic acid dehydrogenase, (5) an .alpha.-glucosidase method comprising decomposing maltose into glucose by .alpha.-glucosidase and assaying the glucose, etc. These enzymatic methods utilize the specificity of enzyme for substrates, and thus have such an advantage as no substantial susceptibility to the influence of the assay-interfering substances, as compared with said methods (1) to (3), but have such disadvantages as a prolonged assay time, an impossibility to assay the whole blood, use of expensive analytical reagents such as enzymes and coenzymes, complicated structures of analytical instruments.
To improve the assaying accuracy and simplify the operating procedure, an enzyme sensor method for assaying amylase has been recently proposed [K. Yoda and T. Tsuchida: Proceedings of the International Meeting on Chemical Sensors, page 648 (1983)]. The principle of the method can be outlined by the following enzymatic reactions. ##STR1##
That is, when oxidation reaction of glucose is carried out with glucose oxidase as an enzyme in the case of assaying glucose, oxygen O.sub.2 is consumed to produce hydrogen peroxide H.sub.2 O.sub.2 according to said equation (III). Different from glucose, said oxygen or hydrogen peroxide can be a target of electrochemical measurement, and thus a glucose concentration can be indirectly measured by electrochemically measuring a decrease in the oxygen amount due to its consumption or an amount of hydrogen peroxide thus produced. In assaying maltose, .alpha.-maltose produced from the saccharides (substrate) by decomposition under the action of amylase contained in the sample according to said equation (I) reacts with water under the action of enzyme .alpha.-glucosidase to produce two molecules of glucose according to said equation (II), and the glucose reacts with water and oxygen under the action of enzyme glucose oxidase to produce gluconic acid and hydrogen peroxide according to said equation (III). In this case, the amount of glucose can be indirectly measured by electrochemically measuring an amount of hydrogen peroxide thus produced, or an amount of oxygen thus decreased in the same manner as described above referring to the assaying of glucose. However, the amount of glucose to be measured in this case is the total amount of the glucose derived from .alpha.-maltose produced from the substrate by decomposition by amylase and the glucose existing in the sample from the initial. Amylase can be assayed from a difference of an output signal obtained by assaying maltose corresponding to the total amount of glucose from an output signal corresponding to the initial glucose amount.
An enzyme electrode for the enzyme sensor method comprises an immobilized enzyme membrane in which glucose oxidase and .alpha.-glucosidase are immobilized, and a transducer capable of electrochemically measuring a change in chemical reaction, occasioned by catalytic actions of these enzymes. The enzyme sensor method is much better than the conventional methods because of higher assay accuracy, shorter assay time, simple analytical instruments, and no requirements for analytical reagents such as coloring reagents, etc. and thus is a very promissing one. However, its effect cannot be fully attained so long as the conventional transducer is used. That is, a galvanic type oxygen electrode, which will be hereinafter referred to as "O.sub.2 electrode", and a polarographic type hydrogen peroxide electrode, which will be hereinafter referred to as "H.sub.2 O.sub.2 electrode", are usually used as the transducer, and the H.sub.2 O.sub.2 electrode is better as a transducer than the O.sub.2 electrode, because the H.sub.2 O.sub.2 electrode that detects the increasing H.sub.2 O.sub.2 has a higher signal/noise ratio and a higher stability in the reaction according to said equation (III) than the O.sub.2 electrode that detects the decreasing O.sub.2. Thus, the H.sub.2 O.sub.2 electrode is preferable as the transducer.
Generally, a H.sub.2 O.sub.2 electrode comprises a gold or platinum anode and a silver cathode. A maltose sensor has an enzyme membrane having the immobilized .alpha.-glucosidase and glucose oxidase, as described above, on the working surface of said electrode. When the ordinary H.sub.2 O.sub.2 electrode having a silver cathode is used, it has been found that a very small amount of silver is dissolved out of the cathode to deactivate the immobilized enzymes, particularly .beta.-glucosidase, in the enzyme membrane.
.alpha.-Glucosidase is deactivated within a few hours even in the immobilized state, whenever it is placed in an atmosphere containing Ag.sup.+ at a concentration of about 10.sup.-5 gram-equivalent/l. On the other hand, the concentration of Ag.sup.+ dissolvable from H.sub.2 O.sub.2 electrode is 10.sup.-5 to 10.sup.-6 gram-equivalent/l at room temperature. Thus, the life of a maltose sensor comprising an H.sub.2 O.sub.2 electrode provided with a silver cathode is one day as the longest.